Multilab evaluation of delafloxacin MIC Test Strip against Gram-negative and Gram-positive organisms.

The performance of the delafloxacin MIC Test Strip (MTS) was evaluated. Three testing sites collected/tested clinical isolates, and 1 site tested challenge isolates that together total 224 S. aureus, 36 S. haemolyticus, 23 S. lugdunensis, 105 E. faecalis, 308 Enterobacteriales, and 140 P. aeruginosa. MIC testing was performed by broth microdilution (BMD) and MTS. Each site also tested 20 common isolates in triplicate on 3 days by MTS and 20 replicates of 4 QC strains by MTS and BMD. MTS results for consolidated clinical/challenge isolates were within 1 doubling dilution of the BMD MIC for 96.9% of S. aureus; 100% of S. haemolyticus, S. lugdunensis, and E. faecalis; 98.4% of Enterobacteriales; and 97.9% of P. aeruginosa. All reproducibility results were within 1 dilution of the modal MIC. All BMD and MTS results for the QC strains were within expected ranges. Overall, the delafloxacin MTS performed similar to BMD.

Multicenter Evaluation of BD Max Enteric Parasite Real-Time PCR Assay for Detection of Giardia duodenalis, Cryptosporidium hominis, Cryptosporidium parvum, and Entamoeba histolytica.

Common causes of chronic diarrhea among travelers worldwide include protozoan parasites. The majority of parasitic infections are caused by Giardia duodenalis, Entamoeba histolytica, Cryptosporidium parvum, and Cryptosporidium hominis Similarly, these species cause the majority of parasitic diarrhea acquired in the United States. Detection of parasites by gold standard microscopic methods is time-consuming and requires considerable expertise; enzyme immunoassays and direct fluorescent-antibody (DFA) stains have lowered hands-on time for testing, but improvements in sensitivity and technical time may be possible with a PCR assay. We performed a clinical evaluation of a multiplex PCR panel, the enteric parasite panel (EPP), for the detection of these common parasites using the BD Max instrument, which performs automated extraction and amplification. A total of 2,495 compliant specimens were enrolled, including 2,104 (84%) specimens collected prospectively and 391 (16%) specimens collected retrospectively. Approximately equal numbers were received in 10% formalin (1,273 specimens) and unpreserved (1,222 specimens). The results from the EPP were compared to those from alternate PCR and bidirectional sequencing (APCR), as well as DFA (G. duodenalis and C. parvum or C. hominis) or trichrome stain (E. histolytica). The sensitivity and specificity for prospective and retrospective specimens combined were 98.2% and 99.5% for G. duodenalis, 95.5% and 99.6 for C. parvum or C. hominis, and 100% and 100% for E. histolytica, respectively. The performance of the FDA-approved BD Max EPP compared well to the reference methods and may be an appropriate substitute for microscopic examination or immunoassays.

Multicenter evaluation of the BD max enteric bacterial panel PCR assay for rapid detection of Salmonella spp., Shigella spp., Campylobacter spp. (C. jejuni and C. coli), and Shiga toxin 1 and 2 genes.

Diarrhea due to enteric bacterial pathogens causes significant morbidity and mortality in the United States and worldwide. However, bacterial pathogens may be infrequently identified. Currently, culture and enzyme immunoassays (EIAs) are the primary methods used by clinical laboratories to detect enteric bacterial pathogens. We conducted a multicenter evaluation of the BD Max enteric bacterial panel (EBP) PCR assay in comparison to culture for the detection of Salmonella spp., Shigella spp., Campylobacter jejuni, and Campylobacter coli and an EIA for Shiga toxins 1 and 2. A total of 4,242 preserved or unpreserved stool specimens, including 3,457 specimens collected prospectively and 785 frozen, retrospective samples, were evaluated. Compared to culture or EIA, the positive percent agreement (PPA) and negative percent agreement (NPA) values for the BD Max EBP assay for all specimens combined were as follows: 97.1% and 99.2% for Salmonella spp., 99.1% and 99.7% for Shigella spp., 97.2% and 98.4% for C. jejuni and C. coli, and 97.4% and 99.3% for Shiga toxins, respectively. Discrepant results for prospective samples were resolved with alternate PCR assays and bidirectional sequencing of amplicons. Following discrepant analysis, PPA and NPA values were as follows: 97.3% and 99.8% for Salmonella spp., 99.2% and 100% for Shigella spp., 97.5% and 99.0% for C. jejuni and C. coli, and 100% and 99.7% for Shiga toxins, respectively. No differences in detection were observed for samples preserved in Cary-Blair medium and unpreserved samples. In this large, multicenter study, the BD Max EBP assay showed superior sensitivity compared to conventional methods and excellent specificity for the detection of enteric bacterial pathogens in stool specimens.

Pediatric multicenter evaluation of the Verigene gram-negative blood culture test for rapid detection of inpatient bacteremia involving gram-negative organisms, extended-spectrum beta-lactamases, and carbapenemases.

We evaluated the investigational use only (IUO) version of the rapid Verigene Gram-negative blood culture test (BC-GN), a microarray that detects 9 genus/species targets (Acinetobacter spp., Citrobacter spp., Enterobacter spp., Escherichia coli/Shigella spp., Klebsiella oxytoca, Klebsiella pneumoniae, Proteus spp., Pseudomonas aeruginosa, and Serratia marcescens) and 6 antimicrobial resistance determinants (blaCTX-M, blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA) directly from positive blood cultures. BC-GN was performed on positive BacT/Alert Pediatric FAN and Bactec Peds Plus blood cultures with Gram-negative organisms at two tertiary pediatric centers. Vitek MS (bioMérieux, Durham, NC) was used to assign gold standard organism identification. The Check MDR CT-102 microarray (Check Points B.V., Wageningen, Netherlands) was used as an alternative method for detecting resistance determinants. In total, 104 organisms were isolated from 97 clinical blood cultures. BC-GN correctly detected 26/26 cultures with Acinetobacter spp., P. aeruginosa, and S. marcescens, 5/6 with Citrobacter spp., 13/14 with Enterobacter spp., 23/24 with E. coli, 2/3 with K. oxytoca, 16/17 with K. pneumoniae, and 0/1 with Proteus spp. BC-GN appropriately reported negative BC-GN results in 8/13 blood cultures that grew organisms that were not represented on the microarray but failed to detect targets in 3/5 cultures that grew multiple Gram-negative organisms. BC-GN detected 5/5 and 1/1 clinical blood cultures with blaCTX-M and blaVIM. All 6 results were corroborated by Check MDR CT-102 microarray testing. The Verigene BC-GN test has the potential to expedite therapeutic decision making in pediatric patients with Gram-negative bacteremia. Sensitivity was satisfactory but may be suboptimal in mixed Gram-negative blood cultures.

The growth and survivability of Streptococcus pneumoniae clinical isolates subjected to various environmental conditions.

Historically, it has been hypothesized that environmental stress would favor the survival of antibiotic susceptible bacteria over resistant ones; however, there is little direct evidence to support this theory. Clinical isolates of S. pneumoniae were chosen and categorized as: penicillin susceptible, quinolone susceptible (PSQS, n = 3); penicillin resistant, quinolone susceptible (PRQS, n = 3); and penicillin resistant, quinolone resistant (PRQR, n = 5). Baseline growth of each isolate was measured by optical density for 24 h. The resulting optical density curves were compared to those obtained for the same isolates subjected to changes in environmental conditions, such as various temperature, pH, and diluted media. In addition, each isolate was inoculated onto cotton fiber disks, held at room temperature, and the recoverable CFU measured over 144 h. In comparison to controls grown under ideal conditions, the density of PSQS isolates was significantly lower than PRQR isolates after 24 h for the following conditions (p < 0.01): incubation at 40 degrees C (1.3 log10 lower); at pH 6.5 (1.6 log10 lower); and in limited nutrient conditions (1.36 log10 lower). When inoculated onto cotton fiber disks, the PRQR isolates decreased an average of 5.0 log10 after 72 h as compared to controls. In contrast, PSQS isolates decreased an average of 8.1 log10 (p < 0.01). Results of this study support the concept that antibiotic resistant isolates may not be at a competitive disadvantage in comparison to susceptible isolates when subjected to some adverse environmental conditions.

Pyogenic arthritis caused by capnocytophaga gingivalis in an immunocompetent three-year-old male.

Capnocytophaga gingivalis is most often isolated as normal oral flora or with periodontal disease. This organism is also associated with sepsis usually in immunocompromised hosts. We identified pyogenic arthritis caused by C. gingivalis in a 3-year-old immunocompetent male, whose clinical course closely resembled monoarticular onset pauciarticular juvenile rheumatoid arthritis. This is the first report of C. gingivalis septic arthritis in the world literature, but there are increasing reports of infections with this carbon dioxide-loving organism at other sites in non-immunocompromised individuals. The subacute presentation of the monoarthritis with this organism of low virulence led to a long delay in diagnosis and treatment. Any monoarthritis must continue to raise concern about infection.

In vitro activity of gemifloxacin and other antimicrobial agents against isolates of Bordetella pertussis and Bordetella parapertussis.

We investigated the activity of the novel quinolone agent gemifloxacin (SB-265805) and a panel of comparator agents against Bordetella pertussis and Bordetella parapertussis. Erythromycin, azithromycin, ciprofloxacin and gemifloxacin were consistently active against both species. An azithromycin- and erythromycin-resistant B. pertussis isolate was not resistant to any of the other agents tested (gemifloxacin MIC < or =0.008 mg/L; ciprofloxacin, 0.015 mg/L; ampicillin, 2.0 mg/L; trimethoprim-sulphamethoxazole, 4.0 mg/L). The potency of ampicillin, azithromycin, erythromycin, ciprofloxacin and trimethoprim-sulphamethoxazole recorded against B. pertussis and B. parapertussis in this study was comparable to that noted in previous studies. However, MICs were generally higher than those noted in other trials; this may reflect the different methods used. Although in vitro data on the potency of gemifloxacin against B. pertussis and B. parapertussis have not previously been reported, these results are comparable to the potency of other quinolones against these pathogens. Should gemifloxacin achieve similar concentrations within the respiratory tract as other quinolones, this, coupled with its high in vitro potency, suggests that gemifloxacin has potential clinical efficacy in pertussis.

The effects of macrolides on the expression of bacterial virulence mechanisms.

Macrolides are unique among the various classes of antimicrobial agents because of the manner in which they interact with pathogens and the host immune system to produce a clinical response. Conventional efficacy explanations require the serum or tissue concentration of the macrolide to exceed the MIC of the macrolide to account for bacteriostatic activity. However, at concentrations below the MIC, there are other effects on the expression of proteinaceous virulence factors which could prevent the pathogen from establishing or expanding an infection. The purpose of this review is to describe these effects and to provide an in-vivo scenario delineating the role of macrolides and leucocytes as they contribute to clinical efficacy.

Molecular epidemiology by ribotyping and PCR-ribotyping of Enterococcus faecium strains isolated from intercontinental areas.

In this study classical ribotyping based on hybridization of an enteroccocal ribosomal operon previously cloned from Enterococcus hirae (Sechi and Daneo-Moore, 1993) with XbaI cut chromosomal DNA and PCR-ribotyping were used to characterize the molecular epidemiology of 131 Enterococcus faecium, with high-level resistance to gentamicin, isolated from different hospitals in Italy and the United States. The ribotyping was able to differentiate all 131 clinical isolates into 96 family patterns. These family patterns appeared to be useful in establishing epidemiological spread. The results obtained were in agreement with those previously published, suggesting the presence of five to six operons in the Enterococcus genus (Sechi et al., 1994). We performed PCR-ribotyping, based on conserved sequences at the 3' end of the enterococcal 16S rrn and the 5' end of the 23S rrn, on 131 clinical isolates as well as on several enterococcal ATCC strains tested. The results were then compared with those obtained with the classical ribotyping method. The results suggest the presence of at least four classes of intergenic spacers among enterococci, but these classes are not helpful in differentiating between Enterococci or among Enterococcal isolates.

The effects of environmental conditions on the in vitro activity of selected antimicrobial agents against Escherichia coli.

Various environmental conditions likely to be encountered at a nidus of infection were evaluated for their effect on selected classes of antimicrobial agents. The minimum inhibitory concentration (MIC) of several aminoglycosides (apramycin, kanamycin, gentamicin, tobramycin, amikacin), tetracycline, and chloramphenicol for five strains of E. coli were unchanged by temperature (35 degrees-39.5 degrees C), atmosphere (aerobic to anaerobic), pH > 7, NaCl concentration (up to 150 mM), zinc concentration (up to 50 mM), and manganese (up to 10 mM). However, the aminoglycoside MICs were increased up to fivefold at pH < 6.5. Magnesium and calcium ion concentrations >10 mM and ferric iron concentrations >/=10 mM increased aminoglycoside MICs from 3.66- to 8-fold. Tetracycline MICs were increased 1.2- to 6.5-fold when the concentration of magnesium or calcium was >/=10 mM. The results of this in vitro study might provide insight into the effects of local in vivo environmental conditions on several classes of antimicrobial agents.

In vitro activity of oral antimicrobial agents against clinical isolates of Pasteurella multocida.

Pasteurella multocida causes a wide variety of infections and is the most common localized soft tissue infection after animal bite injuries. Penicillin or amoxicillin has been considered agent of choice for therapy. Reported beta-lactamase production by some isolates, the therapeutic dilemma of the penicillin allergic patient, and the polymicrobial nature of some infections led to this study of alternate antimicrobial agents. The in vitro activity of ampicillin, amoxicillin/clavulanate, cefprozil, cefuroxime, erythromycin, clarithromycin, trimethoprim/sulfamethoxazole, ciprofloxacin, and tetracycline were compared to penicillin against 73 geographically diverse isolates of P. multocida from human infections collected since 1991. MIC90 (microgram/mL) were as follows: penicillin < or = 0.06; ampicillin < or = 0.5; amoxicillin/clavulanate < or = 0.5; cefaclor 1.0; cefprozil 1.0; cefpodoxime 0.06; cephalothin 2.5; cefuroxime < or = 0.25; erythromycin 2.0; azithromycin 1.0; clarithromycin 4.0; trimethoprim/sulfamethoxazole < or = 0.5/9.5; ciprofloxacin < or 0.25; tetracycline < or = 2.0. No beta-lactamase producing isolates were found in this study. This in vitro study has identified alternate oral agents to penicillins that may be appropriate for therapy of P. multocida infections.

In vitro susceptibility testing of topical antimicrobial agents used in pediatric burn patients: comparison of two methods.

One hundred and seventy-seven bacterial isolates obtained from pediatric burn victims were tested for in vitro susceptibility against bacitracin, silver sulfadiazine, mafenide acetate, nitrofurazone, and mupirocin by two methods: standard microbroth dilution and Nathan's agar well diffusion (NAWD). Nitrofurazone had the broadest spectrum of activity. Mupirocin was the most potent agent against methicillin-susceptible Staphylococcus aureus. Silver sulfadiazine showed activity against gram-positive organisms and higher minimum inhibitory concentration (MIC) values, and smaller zone sizes were seen for methicillin-resistant S. aureus and gram-negative bacilli. Bacitracin showed activity against S. aureus and Streptococcus pyogenes by the microbroth method; activity could not be assessed by NAWD. Mafenide acetate had the highest MICs for all isolates tested. Correlation between methods for all isolates tested was best for mupirocin and nitrofurazone. NAWD was labor intensive and difficult to interpret; MIC method was easy to perform and reproducible. Clinical correlation is necessary to establish breakpoints for interpretation of test results.

Comparison of five different susceptibility test methods for detecting antimicrobial agent resistance among Haemophilus influenzae isolates.

The detection of antimicrobial agent resistance among ninety-eight Haemophilus influenzae isolates was assessed by six different antibiotic test methods: agar dilution on Mueller-Hinton agar supplemented with 5% lysed horse blood (MH-LHB), E-test using both Haemophilus test medium (HTM) agar and chocolate Mueller-Hinton (CMH) agar plates, Vitek Haemophilus susceptibility cards, and three overnight microdilution systems that included two commercial systems, Micro-Media and MicroScan, and the reference broth microdilution method using HTM broth. Agents tested in the study included ampicillin, amoxicillin/clavulanic acid (A/C), cefaclor, cefuroxime, cefotaxime, ceftriaxone, chloramphenicol, and trimethoprim/sulfamethoxazole. Both the reference HTM microbroth dilution method and agar dilution correctly classified all nine of the beta-lactamase negative ampicillin resistant (BLNAR) isolates. Each of the other test methods failed to detect one of the BLNAR strains, either because of growth failure (Micro-Media and MicroScan) or miscategorization of an isolate as susceptible (E-Test HTM, E-Test CMH, and Vitek). None of the test methods detected all six isolates identified as A/C resistant by HTM microbroth dilution. Of the remaining antimicrobials tested, ampicillin and cefuroxime yielded data that could be compared by all test methods. The very major, major, and minor errors for these two antimicrobials in comparison to the reference HTM microdilution method were as follows: Micro-Media (1.7%, 0%, and 4.8%); MicroScan (11.9%, 0%, and 8.1%); E-Test HTM (1.6%, 0%, and 2.0%); E-Test CMH (1.6%, 1.6%, and 4.6%); Vitek (8.1%, 0%, and 3.1%); and agar dilution on MH-LHB (0%, 0%, and 4.6%). Micro-Media and MicroScan panels failed to support the growth of 4.1% and 5.1% of the isolates, respectively.

A novel tetracycline-resistant determinant, tet(U), is encoded on the plasmid pKq10 in Enterococcus faecium.

Nine tetracycline (Tc)-resistant clinical isolates of Enterococcus faecium were screened for plasmid content using agarose gel electrophoresis. pKQ10, a 1.9-kb plasmid carrying a novel Tc resistance determinant, was isolated from one of the isolates. The nucleotide sequence of this plasmid revealed an open reading frame corresponding to an 11.8-kDa protein and containing 105 amino acid residues. There was some limited similarity between this protein and tet(M), tet(O), tet(Q), tet(S), tetB(P), and otr(A), which overlapped, but did not include, the consensus GTP-binding sequences. The low-level, Tc-resistant determinant of pKQ10, named tet(U), does not appear to correspond to any other known Tc resistance determinant.

Comparative in vitro activity of furopenem against aerobic bacteria isolated from pediatric patients.

The in vitro activity of the oral penem furopenem (WY-49605, 545555, SUN5555, and ALP201) was tested against clinical bacteria isolated from pediatric patients. Furopenem was compared with clarithromycin, cefpodoxime, amoxicillin, amoxicillin-clavulanate, cefaclor, cefixime, and cefuroxime. Furopenem demonstrated consistent activity against Escherichia coli [minimum inhibitory concentration (MIC90) = 1.0 microgram/ml)] Klebsiella pneumoniae (MIC90 = 2.0 micrograms/ml), Salmonella enteriditis and Shigella spp. (MIC90 = 1.0 microgram/ml), and beta-lactamase-positive or -negative Haemophilus influenzae (MIC90 = 1.0 microgram/ml) and Moraella catarrhalis (MIC90 = 1.0 microgram/ml). Furopenem was also active against a number of the Gram-positive organisms tested including methicillin-susceptible Staphylococcus aureus and penicillin-susceptible Streptococcus pneumoniae. These results suggest a potential application for this agent in the treatment of children as outpatients.

Recovery of Pseudomonas cepacia and other Pseudomonas species from the environment.

Nine hundred sixteen cultures were obtained from homes of patients with cystic fibrosis, control homes, salad bars, and food markets, and analyzed for the presence of Pseudomonas cepacia and related bacteria. P cepacia was recovered from 5 (18%) of 27 homes, and from 20 (4%) of 509 cultures collected outside of homes. Relative to other pseudomonads, P cepacia is found infrequently in the environment. It is not clear how frequently these sources contribute to acquisition of this bacteria by persons with cystic fibrosis.